Peter Terness

Role of human corneal endothelial cells in T-cell-mediated alloimmune attack in vitro.

PURPOSE Human corneal endothelial cells (HCEC) are a potential target of immune attack after corneal transplantation. The aim of this in vitro study was to investigate the role of HCEC during the alloimmune response of T-cells by examining cytokine profiles, function of the immunosuppressive enzyme indoleamine 2,3-dioxigenase (IDO), major histocompatibility complex (MHC-I/-II), T-cell proliferation, and the induction of cell death. METHODS Real-time PCR and RP-HPLC were used to determine IDO expression and activity. Multiplex assay was performed for quantification of cytokine levels. T-cell proliferation was assessed by thymidine incorporation, and HCEC cell death was measured by flow cytometry. RESULTS Human corneal endothelial cells induce strong proliferation of allogeneic T-cells and an increase of proinflammatory cytokines such as interleukin-1α (IL-1α), IL-1β, IL-6, interferon-gamma (IFN-γ), and tumor necrosis factor-alpha (TNF-α). Tumor necrosis factor-alpha (and to a lesser extent IFN-γ) induces apoptosis. Moreover, IFN-γ strongly upregulates MHC-II molecules and IDO activity in HCEC as reflected by high kynurenine (Kyn) concentrations. Interestingly, the T-cell response was not affected by increased IDO activity, since blocking of IDO did not affect the proliferation rate. Indoleamine 2,3-dioxigenase-induced Kyn levels did not exceed concentrations of 175 ± 20 μM. Concentrations of ≥400 μM Kyn were required to suppress T-cell proliferation. CONCLUSIONS Our data show that T-cell attack on HCEC leads to increased concentrations of proinflammatory cytokines. Inflammatory cytokines induce apoptosis and upregulate MHC-II molecules and IDO in HCEC. Although increased IDO activity does not influence the T-cell response, it constitutes an inflammatory marker of the alloimmune response toward HCEC.

Short-term T cell receptor directed immunotherapy induces organ specific peripheral tolerance in a strongly incompatible rat model

We analysed the effect of selective alpha/beta-T cell elimination on allograft survival in a strongly histoincompatible DA-->LEWIS rat model by treatment of recipients with the mouse monoclonal antibody R73 two times before and seven times after transplantation. R73 induced virtually indefinite cardiac allograft survival in 44% of six-week-old LEWIS recipients, whereas donor-type skin allografts were rejected within 11 days. The remaining 56% of animals presented a mean cardiac survival time of 41 +/- 13 days. Graft prolongation was age dependent since in ten-week-old animals the survival time was only of 19 +/- 5 days (untreated controls: 7 +/- 1 days). R73 induced a rapid decrease of R73-positive T cells in the peripheral blood from 70% before treatment to 2%. From the fifth day of treatment a gradual T cell recovery was registered. The T cell marker CD5 decreased from 72% to 17% but recovered already from the second day of treatment. Determination of alpha/beta-TCR, CD3 and CD5 density on T cells during R73 therapy showed that the initial T cell decrease was due to T cell elimination, whereas modulation of alpha/beta-TCR was predominant during the following days. Anti-R73 antibodies appeared regularly during the first week of treatment and blocked R73 activity, indicating their anti-idiotypic nature. The present findings show that short-term R73 therapy is able to induce long-lasting allograft survival. This experimental model can be used to study the basis of peripheral organ specific T cell tolerance.